* Janet had it crash by trying to press save while it was reading a time course.

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* Things that cause it to crash:

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* Amanda had it freeze because the file was "too big" 12/2013. It was 1.3 MB, and froze upon saving. She gave it 5 min to save, but the program said "not responding" in the force quit box. The file produced after force quitting was broken: the data was gone when she tried to open it again. Amanda has done bigger files before, so this was a little surprising. Customer support said that it can crash if you have files that are too big. Break your runs down into different files if you are worried. Amanda also suggests giving it as long as possible; maybe it would have come back to life.

** Making a new plate while data is being recorded, if the plate selected when you click to make a new plat is the plate being recorded.

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** Amanda had tried the autosave option but it was saving files in unusual locations.

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** Having files that are "too big."

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** Do not save while the software is trying to do something else (like read a plate).

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*** Amanda had it freeze because the file was "too big" 12/2013. It was 1.3 MB, and froze upon saving. She gave it 5 min to save, but the program said "not responding" in the force quit box. The file produced after force quitting was broken: the data was gone when she tried to open it again. Amanda has done bigger files before, so this was a little surprising. Customer support said that it can crash if you have files that are too big. Break your runs down into different files if you are worried. Amanda also suggests giving it as long as possible; maybe it would have come back to life.

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* Ways to use the software with minimal trauma:

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** Save often

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*** Save after each strip or chunk of strips you monitor.

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*** Pressing save can be slow, but "save as" is much faster. Make many iterations of a file: file-revA.pda ... file-revK.pda

Making a new plate while data is being recorded, if the plate selected when you click to make a new plat is the plate being recorded.

Having files that are "too big."

Amanda had it freeze because the file was "too big" 12/2013. It was 1.3 MB, and froze upon saving. She gave it 5 min to save, but the program said "not responding" in the force quit box. The file produced after force quitting was broken: the data was gone when she tried to open it again. Amanda has done bigger files before, so this was a little surprising. Customer support said that it can crash if you have files that are too big. Break your runs down into different files if you are worried. Amanda also suggests giving it as long as possible; maybe it would have come back to life.

Ways to use the software with minimal trauma:

Save often

Save after each strip or chunk of strips you monitor.

Pressing save can be slow, but "save as" is much faster. Make many iterations of a file: file-revA.pda ... file-revK.pda

How much of my substance can I add?

Note how wobbly the lines are, even though the sample was shaken for 20 seconds by the machine before reading began.

mM NADH on Molecular Devices and OD340

Order of reagents

Example 1:

add 20-40uL cell extract per well. Add ~180 uL of mix of substrate & cofactors dissolved in buffer. Adding the large volume to the small volume should yield decent mixing.

Should I run a whole plate at once or rows/columns at a time?

There are a few reasons you want to read the wells you load as quickly as possible. As soon as you mix all your reagents and cell extract, your reaction starts. If your assay has a non-linear reaction rate at short times (often the case) then you will lose some information at early timepoints. For example, often a reaction is fastest at small times, and slows down as the reaction proceeds and products build up.

Unless you know your assay is highly linear for 30+ seconds, Janet recommends you run one cell extract with every substrate/no-substrate mix per scan of the machine, then move to the next one. OR, run all of your cell extracts with one substrate/no-substrate mix at a time.

Do I need to use the crystal plate or a plastic plate?

NADH assays are run at 340 nm. This is right on the lower boundary for what wavelengths are suitable for our plastic so it shouldn't matter much. If the crystal plate is around, you might as well use it. At a minimum you will generate less trash.

Exporting Data

If kinetic data needs to be exported and you can't get the data exported as one column per well:

Go to Settings --> Preferences

Change the settings to Time

Your files should look like this:

best export format for Softmax Pro-kinetic data

Note: This assay ran for < 1 minute and Microsoft excel misinterpreted the times as minutes, not seconds. This may not be a problem if you run your assay longer, but it was a problem here.

Kinetic Data

The machine can report Vmax in units of milli-Units/min or Units/second. What are "Units" you ask? It is just the slope. Multiply by 1,000 to get d(Absorbance)/min.

Plates

What type to use when detecting NAD/NADH

You can chose between the crystal plate and disposable plastic plates. The decision is a trade-off between getting (possibly) better optics and less interferance at 340nm with a crystal plate, however the crystal plate builds up residue that may lead to inaccurate assay results.

Use diluted alkaline (0.1 M NaOH) or diluted acid (0.1 M HCl) solutions, followed by several washes with deionized water. You can also use a neutral pH detergent in warm water, followed by the 0.1 M HCl wash and deionized water rinses

Do not use hydrofluoric acid, as it can attack the quartz material

To avoid scratches, use only lens cleaning tissues or very fine cloth to wipe the surface of the cuvette window

Using an ultrasonic cleaner is not recommended, as it may cause the cuvette to break